Cyclosporin analog formulations

ABSTRACT

The present disclosure relates to formulations containing cyclosporin analogs that are structurally similar to cyclosporin A, in particular isomeric mixtures of cyclosporin analogs that are structurally similar to cyclosporin A. The formulations form stable microemulsion preconcentrates and may provide superior drug bioavailability and/or may reduce one or more adverse effects associated with the administration of cyclosporin. Also disclosed are methods for using and preparing the formulations.

CLAIM OF PRIORITY

This application is a continuation of U.S. application Ser. No.12/941,907, filed Nov. 8, 2010, which application is a continuation ofU.S. application Ser. No. 12/197,199, filed Aug. 22, 2008, issued asU.S. Pat. No. 7,829,533, which application is a divisional of U.S.application Ser. No. 11/375,532, filed Mar. 13, 2006, now U.S. Pat. No.7,429,562, which is a continuation of U.S. application Ser. No.10/274,419, filed Oct. 17, 2002, now U.S. Pat. No. 7,060,672, issuedJun. 13, 2006, which claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 60/370,597 filed on Apr. 5, 2002, andto U.S. Provisional Application Ser. No. 60/346,201 filed on Oct. 19,2001, the disclosures of all of which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to formulations containing cyclosporinanalogs that are structurally similar to cyclosporin A. In particular,the formulations contain isomeric mixtures of the cyclosporin analogISA_(Tx)247. These formulations form stable microemulsions which providehigh drug solubility, superior drug bioavailability and may reduce oneor more adverse effects associated with the administration ofcyclosporin. Also disclosed are methods for using and preparing theformulations.

BACKGROUND OF THE INVENTION References

The following references are referred to by numbers in parentheses atthe relevant portion of the specification.

-   1. Gupta and Robinson, Treatise on Oral Controlled-drug Delivery.    Text Ed. 1992, edited by Aegis Cadence, Mandel Decker, Inc.-   2. Traber et al., Helv. Chim. Acta, 60: 1247-1255 (1977)-   3. Kobel et al., Europ. J. Applied Microbiology and Biotechnology,    14: 237-240 (1982)-   4. von Wartburg et al, Progress in Allergy, 38: 28-45 (1986)-   5. Rich et al. (J. Med. Chem., 29: 978 (1986)-   6. U.S. Pat. No. 4,384,996, issued on May 24, (1983)-   7. U.S. Pat. No. 4,771,122, issued on September 13, (1988)-   8. U.S. Pat. No. 5,284,826, issued on February 8, (1994)-   9. U.S. Pat. No. 5,525,590, issued on June 11, (1996)-   10. Sketris et al., Clin. Biochem., 28: 195-211 (1995)-   11. Bennett, Renal Failure, 20: 687-90 (1998)-   12. Wang et al., Transplantation, 58:940-946 (1994)-   13. “Eastman Vitamin E TPGS”, Eastman Brochure, Eastman Chemical    Co., Kingsport, Term. (October 1996)-   14. Rowley's Condensed Chemical Dictionary, (1987)-   15. Ellis, Progress in Medicinal Chemistry 25, (1988) Elsevier,    Amsterdam-   16. Sokol, R. J., Lancet, 338(8761): 212, (1991)-   17. Sokol, R. J., Lancet, 338(8768): 697, (1991)

The disclosure of each of the above publications or patents is herebyincorporated by reference in its entirety to the same extent as if thelanguage of each individual publication and patent were specifically andindividually incorporated by reference.

The Use of Cyclosporin as a Therapeutic Agent

Despite efforts to avoid graft rejection through host-donor tissue typematching, in the majority of transplantation procedures,immunosuppressive therapy is critical to the viability of the donororgan in the host. A variety of immunosuppressive agents have beenemployed in transplantation procedures, including azathioprine,methotrexate, cyclophosphamide, FK-506, rapamycin and corticosteroids.Cyclosporins are finding increasing use in immunosuppressive therapy dueto their preferential effect on T-cell mediated reactions (1).

Cyclosporin is a potent immunosuppressive agent that has beendemonstrated to suppress humoral immunity and cell-mediated immunereactions such as allograft rejection, delayed hypersensitivity,experimental allergic encephalomyelitis, Freund's adjuvant arthritis andgraft versus host disease (GVHD). It is used for the prophylaxis oforgan rejection subsequent to organ transplantation, for treatment ofrheumatoid arthritis, for the treatment of psoriasis and for thetreatment of other autoimmune diseases such as type I diabetes, Crohn'sdisease and lupus. Many naturally occurring cyclosporins are well knownin the art. Non-natural cyclosporins have been prepared by total- orsemi-synthetic means or by the application of modified culturetechniques. Thus, the class of available cyclosporins is substantial,and includes, for example, the naturally occurring cyclosporins A (CsA)through Z (CsZ), as well as other non-natural cyclosporin derivativessuch as dihydro- and iso-cyclosporins (2, 3, 4). CsA analogs containingmodified amino acids in the 1-position have been reported by Rich et al.(5). Immunosuppressive, anti-inflammatory and anti-parasitic CsA analogsare described in U.S. Pat. Nos. 4,384,996 (6), 4,771,122 (7), 5,284,826(8) and 5,525,590 (9), assigned to Sandoz.

Cyclosporin is a lipophilic molecule having a molecular weight of 1202daltons. Owing to the poor solubility in water and the highlipophilicity of cyclosporin A, pharmaceutical compositions ofcyclosporin A with customary solid or liquid pharmaceutical carriersoften have disadvantages. For example, the cyclosporins are notsatisfactorily absorbed from such compositions, or the compositions arenot well tolerated, or they are not sufficiently stable when stored.Often, the dissolved concentration is low in relation to the daily dose.

Adverse Effects of Cyclosporin Therapy

There are numerous adverse effects associated with cyclosporin therapy.These include nephrotoxicity, hepatotoxicity, cataractogenesis,hirsutism, parathesis and gingival hyperplasia (10). The most seriousadverse effect is nephrotoxicity.

Acute cyclosporin nephrotoxicity is accompanied morphologically bytubular lesions characterized by inclusion bodies, isometric vacuolationand microcalcification. That leads to a decrease in the glomerularfiltration rate, which can be identified by the rapid increase in serumcreatinine in patients treated with cyclosporin (11).

The exact mechanism by which cyclosporin causes renal injury is notknown. In rat studies, chronic CsA-induced functional and structuraldeterioration of the kidney was accompanied by renal lipid peroxidation.It has been shown by Wang et al. that co-administration of anantioxidant with CsA reduced the renal injury in the rat (12).

Previous attempts in the art to reduce the nephrotoxic risks associatedwith cyclosporin therapy include the co-administration of the drug withan agent that delays the metabolism of cyclosporin, effectively reducingthe dose required to maintain therapeutic blood levels. However, thismethod often does not resolve the problem of high variation ofcyclosporin bioavailability(12). Thus, the problem of preparing acyclosporin formulation that has excellent bioavailability but whichdoes not cause adverse side effects is well known in the art.

Dosage Forms

In order for a drug to perform its therapeutic activity, a therapeuticamount of the drug must be made bioavailable, i.e., it must be able toget to the site of action in the patient. Oral drug delivery is known inthe art, and popular because of ease of administration. However, oraldosage forms are complicated by the fact that the drug must first bereleased from the dosage form over a given time in the gut, and must besolubilized and absorbed in the gastrointestinal tract. Thus, properdrug release from the dosage form and solubilization of the drug in thegastrointestinal (GI) tract are critical.

Well known oral dosage forms that work well within the confines of theGI tract include tablets, capsules, gel capsules, syrups, suspensions,emulsions, microemulsions and pre-emulsion concentrates. Solubilityplays a large role in the development of oral dosage formulations,because the formulation used to deliver the active drug will affect theamount and/or concentration of the drug that reaches the active siteover a given period of time. The composition of the formulation alsodirectly affects the solubilization of the drug compound in thegastrointestinal tract, and consequently the extent and rate of theabsorption of the active drug compound into the blood stream. Inaddition, the therapeutic value of a drug is affected by the rate inwhich the drug is released from the delivery system itself, which inturn affects the rate and extent of solubilization of the activecompound in the gastrointestinal tract before absorption (1).

In conventional systems known in the art, drug content is released intothe gastrointestinal tract within a short period of time, and plasmadrug levels peak at a given time, usually within a few hours afterdosing. The design of known oral drug delivery systems, includingcyclosporin formulations, is based on obtaining the fastest possiblerate of drug dissolution at the risk of creating undesirable, doserelated side effects.

Thus, there is a serious need for cyclosporin formulations with reducedtoxicity but which retain high levels of bioavailability, and which donot need to be administered with another agent.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that certainformulations, preferably microemulsion preconcentrate and microemulsionformulations, can provide the delivery of the cyclosporin analog,ISA_(Tx)247, which reduces one or more of the adverse effects associatedwith cyclosporin, while maintaining a high bioavailability of the drug.Further, the formulations of the present invention also increase theimmunosuppressive effects of ISA_(Tx)247 by providing sufficientbioavailability.

The formulations of the present invention all contain ISA_(Tx)247 as theactive ingredient and synthetic or vegetable oils emulsified with asynthetic co-emulsifier such as a non-ionic surfactant that has apolyoxylated moiety. Thus, the present formulations contain ISA_(Tx)247,a surfactant, ethanol, a lipophilic and/or an amphiphilic solvent. Theformulations of the present invention may be adjusted for pH andisotonicity as needed, and may also include biocompatable polymers suchas protective colloids, suspension stabilizers and building agents,excipients, binders and carriers, as needed.

In one preferred aspect, the invention is directed to a microemulsionpreconcentrate comprising a) a pharmaceutically effective amount ofISA_(Tx)247, b) Vitamin E TPGS, c) MCT oil, d) an emulsifier selectedfrom the group consisting of Tween 40 and Tween 80; and e) ethanol.Preferably the microemulsion preconcentrate comprises from about 5 toabout 10% by weight of ISA_(Tx)247, greater than 0% to about 50% byweight of Vitamin E TPGS, greater than 0% to about 50% by weight of MCToil, greater than 0% to about 50% by weight of emulsifier and about 5%to about 15% by weight of ethanol, wherein the formulation is a liquidmicroemulsion preconcentrate at room temperature. More preferably, theratio by weight of a:b:c:d:e is about 0.5-1:4:2:2:1.

The present invention also contemplates a method of preparing amicroemulsion preconcentrate comprising mixing the MCT oil, theemulsifier, the ethanol, the Vitamin E TPGS, and the proper amount ofISA_(Tx)247 until the ISA_(Tx)247 is completely dissolved, wherein theformulation is a liquid at room temperature.

In another aspect, the invention is directed to a pharmaceuticalformulation comprising a) a pharmaceutically effective amount ofISA_(Tx)247, b) Vitamin E d-a-tocopheryl polyethylene glycol 1000succinate (Vitamin E TPGS), c) medium chain triglyceride (MCT) oil, d)Tween 40, and e) ethanol. Preferably the pharmaceutical formulation is amicroemulsion; more preferably the formulation is a microemulsionpreconcentrate. The formulation preferably comprises from about 5 toabout 10% by weight of ISA_(Tx)247, from about 20 to about 50% by weightof Vitamin E TPGS, from about 5 to about 20% by weight of MCT oil, fromabout 5% to about 50% of Tween 40 and about 5 to about 15% by weight ofethanol. More preferably, the ratio by weight of a:b:c:d:e is about0.5-1:4:2:2:1.

In a further aspect, the invention is directed to a method of producingimmunosuppression comprising administering to a subject in need thereofan effective amount of the pharmaceutical formulation comprising a) apharmaceutically effective amount of ISA_(Tx)247, b) Vitamin E TPGS, c)MCT oil, d) Tween 40, and e) ethanol.

The present invention also contemplates a method of preparing thepharmaceutical formulation of the present aspect of the inventioncomprising mixing the MCT oil, the Tween 40, the ethanol, the Vitamin ETPGS, and the proper amount of ISA_(Tx)247 until the ISA_(Tx)247 iscompletely dissolved.

In a further aspect, the invention is directed to a pharmaceuticalformulation comprising a) a pharmaceutically effective amount ofISA_(Tx)247, b) Vitamin E TPGS, c) MCT oil, d) Tween 80, and e) ethanol.Preferably the pharmaceutical formulation is a microemulsion; morepreferably the formulation is a microemulsion preconcentrate. Thepharmaceutical formulation preferably comprises from about 5% to about10% by weight of ISA_(Tx)247, from about 20% to about 50% by weight ofVitamin E TPGS, from about 5% to about 20% by weight of MCT oil, fromabout 5% to about 50% by weight of Tween 80 and about 5% to about 15% byweight of ethanol. More preferably, the ratio by weight of a:b:c:d:e isabout 0.5-1:2:4:2:1.

In a further aspect, the invention is directed to a method of producingimmunosuppression comprising administering to a subject in need thereofan effective amount of the pharmaceutical formulation comprising a) apharmaceutically effective amount of ISA_(Tx)247, b) Vitamin E TPGS, c)MCT oil, d) Tween 80, and e) ethanol.

The present invention also contemplates a method of preparing thepharmaceutical formulation of the present aspect of the inventioncomprising mixing the MCT oil, the Tween 80, the ethanol, the Vitamin ETPGS, and the proper amount of ISA_(Tx)247 until the ISA_(Tx)247 iscompletely dissolved.

In yet a further aspect, the present invention is directed to apharmaceutical formulation comprising a) a pharmaceutically effectiveamount of ISA_(Tx)247, b) MCT oil, c) Tween 80, d) triacetin, and e)ethanol. Preferably the pharmaceutical formulation is a microemulsion;more preferably the formulation is a microemulsion preconcentrate. Thepharmaceutical formulation preferably comprises from about 5% to about10% by weight of ISA_(Tx)247, from about 20% to about 50% by weight oftriacetin, from about 5% to about 50% by weight of MCT oil, from about5% to about 50% by weight of Tween 80 and about 5% to about 15% byweight of ethanol. More preferably, the ratio by weight of a:b:c:d:e isabout 0.5-1:5:3:1:1.

In a further aspect, the invention is directed to a method of producingimmunosuppression comprising administering to a subject in need thereofan effective amount of the pharmaceutical formulation comprising a) apharmaceutically effective amount of ISA_(Tx)247, b) MCT oil, c) Tween80, d) triacetin, and e) ethanol.

The present invention also contemplates a method of preparing thepharmaceutical formulation of the present aspect of the inventioncomprising mixing the MCT oil, the Tween 80, the ethanol, the triacetin,and the proper amount of ISA_(TX)247 until the ISA_(Tx)247 is completelydissolved.

In still a further aspect, the invention is directed to a pharmaceuticalformulation comprising a) a pharmaceutically effective amount of a)ISA_(Tx)247, b) Tween 80, c) Vitamin E TPGS, d) ethanol, and e)isopropyl myristate. Preferably the pharmaceutical formulation is amicroemulsion; more preferably, the formulation is a microemulsionpreconcentrate. The pharmaceutical formulation preferably comprises fromabout 5% to about 10% by weight of ISA_(Tx)247, from about 20% to about50% by weight of Vitamin E TPGS, from about 5% to about 55% by weight ofisopropyl myristate, from about 5% to about 50% by weight of Tween 80and about 5% to about 15% by weight of ethanol. More preferably, theratio by weight of a:b:c:d:e is about 0.5-1:2:1:1:6.

In a further aspect, the invention is directed to a method of producingimmunosuppression comprising administering to a subject in need thereofan effective amount of the pharmaceutical formulation comprising a)ISA_(Tx)247, b) Tween 80, c) Vitamin E TPGS, d) ethanol, and e)isopropyl myristate.

The present invention also contemplates a method of preparing thepharmaceutical formulation of the present aspect of the inventioncomprising mixing the isopropyl myristate, the Tween 80, the ethanol,the Vitamin E TPGS n, and the proper amount of ISA_(Tx)247 until theISA_(Tx)247 is completely dissolved.

In still a further aspect, the invention is directed to a pharmaceuticalformulation preconcentrate comprising a) a pharmaceutically effectiveamount of ISA_(Tx)247, b) Vitamin E TPGS, c) MCT oil, d) Tween 40, ande) ethanol. The pharmaceutical formulation preconcentrate preferablycomprises from about 5% to about 10% by weight of ISA_(Tx)247, fromabout 20% to about 50% by weight of Vitamin E TPGS, from about 5% toabout 20% by weight of MCT oil, from about 5% to about 50% by weight ofTween 40 and about 5% to about 15% by weight of ethanol. Morepreferably, the ratio by weight of a:b:c:d:e is about 0.5:4:2:2:1. Evenmore preferably, the formulation is a microemulsion preconcentrate,wherein the preconcentrate, when mixed with an aqueous media, forms aclear stable microemulsion solution. Preferably, the aqueous media iswater or fruit juice, excluding grapefruit juice. In addition, thepreconcentrate is preferably mixed with the aqueous media in a ratio ofabout 1 part preconcentrate to between about 10 and about 100 partsaqueous media.

In a further aspect, the invention is directed to a method of producingimmunosuppression comprising administering to a subject in need thereofan effective amount of the pharmaceutical formulation comprising a) apharmaceutically effective amount of ISA_(Tx)247, b) Vitamin E TPGS, c)MCT oil, d) Tween 40, and e) ethanol, mixed into an aqueous media.

The present invention also contemplates a method of preparing thepharmaceutical formulation of the present aspect of the inventioncomprising mixing the MCT oil, the Tween 40, the ethanol, the Vitamin ETPGS n, and the proper amount of ISA_(Tx)247 until the ISA_(Tx)247 is'completely dissolved. The resulting preconcentrate is then mixed withaqueous media, preferably in a ratio of about 1 part preconcentrate toabout 10 to about 100 parts media.

In yet another aspect, the invention is directed to a pharmaceuticalformulation comprising: a) a pharmaceutically effective amount ofISA_(Tx)247, b) Vitamin E TPGS, c) MCT oil, d) Tween 40, and e) ethanol.The pharmaceutical formulation may be administered parenterally, such assubcutaneously (SC) or intramuscularly (IM) and is optionallysterilized.

In another aspect of the present invention any one of the microemulsionprecohcentrate formulations maybe mixed with an aqueous media to form aclear thermodynamically stable microemulsion solution. The aqueous mediamay be water or fruit juice, excluding grapefruit juice, which may reactadversely with the ISA_(Tx)247. In addition, the preconcentrate may bemixed with the aqueous media in a ratio of about 1 part preconcentrateto between about 10 and about 100 parts aqueous media.

In yet another aspect of the present invention, any one of themicroemulsion preconcentrate formulations maybe an oral formulation,preferably encapsulated in a soft gelatin capsule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a table for the formation of microemulsion preconcentratesas a function of excipient ratio for Tween 40 and MCT Oil.

FIG. 2 shows a graph providing a comparison of pharmacokinetic (PK)profiles for our ISA_(Tx)247 microemulsion in comparison with Neoral® inBeagle dogs.

FIG. 3 shows an overview of exemplary synthetic pathways that may beused to prepare the cyclosporin analogs of the present invention, wherestereoselective pathways are grouped according to reactive conditions;

FIG. 4 illustrates a synthetic pathway that produces a mixture of (E)and (Z)-isomers of ISA_(Tx)247 from a bromine precursor; and

FIG. 5 illustrates another synthetic pathway that produces a mixture of(E) and (Z)-isomers of ISA_(Tx)247 from an aldehyde precursor.

DETAILED DESCRIPTION OF THE INVENTION

In general, the terms in the present application are used consistentlywith the manner in which those terms are understood in the art.

The terms “cyclosporine” and “cyclosporine” are simply a variation inspelling and are used herein interchangeably to refer to the samecompound, for example cyclosporin A and cyclosporine A are the samecompound) or the same class of compounds, as appropriate.

By the term “microemulsion preconcentrate”, as used herein, is meant asystem capable upon contact with an aqueous media of providing amicroemulsion. The term microemulsion is used herein in itsconventionally accepted sense as a non-opaque (clear) or substantiallynon-opaque thermodynamically stable colloidal dispersion comprisingwater and organic components including hydrophobic (lipophilic) organiccomponents.

The term “emulsion” is a heterogeneous system, consisting of at leastone immiscible liquid intimately dispersed in another in the form ofdroplets, whose diameters, in general, exceed 0.1 mm. Such systemspossess a minimal stability, which may be accentuated by additives suchas surface-active agents, finely divided solids, etc. Conventional“emulsions”, unlike the microemulsions of the present invention, areconsidered to be thermodynamically unstable dispersions.

A. Preparation of Formulations

The formulations of the present invention all contain ISA_(Tx)247 as theactive ingredient and synthetic or vegetable oils emulsified with acoemulsifier, preferably a synthetic co-emulsifier such as a non-ionicsurfactant that has a polyoxylated moiety. The formulations of thepresent invention may be adjusted for pH and isotonicity as needed. Theformulations of the present invention may also include biocompatablepolymers such as protective colloids, suspension stabilizers andbuilding agents, excipients, binders and carriers, as needed. Certainpreferred embodiments form clear stable formulations providingrelatively high drug solubility and relatively quick dissolution inaqueous media (less than about 5 minutes with stirring) to form clearmicroemulsions.

Thus, the present formulation is a microemulsion, or a microemulsionpreconcentrate, containing ISA_(Tx)247, a surfactant, an oil, ethanol,and an emulsifier that is either a lipophilic and/or an amphiphilicsolvent. In certain embodiments, the preparation comprises from about 5%to about 10% by weight of ISA_(Tx)247, from about 20% to about 50% byweight of a surfactant, such as tocopheryl polyethylene glycolcarboxylic acid ester (or, optionally, emulsion stabilizers such astriacetin) from about 5% to 20% by weight of an oil component, such asMCT oils or isopropyl myristate, from about 5% to about 15% by weight ofethanol, and from about 20% to about 40% by weight of a lipophilicsolvent and/or from about 10% to about 55% by weight of an amphiphilicsolvent. In addition, the formulation may optionally contain from about10% to about 20% by weight of another surfactant.

Embodiments of the present invention may have sufficient drugbioavailability (see Tables. 3 and 5 and FIG. 2) and/or decreasedtoxicity (see Tables 5 and 7). The mechanisms for this increasedbioavailability and/or decreased toxicity are unknown. However, withoutbeing limited by theory, Vitamin E TPGS may contribute to these featuresof the formulation. For example, co-administration of Vitamin E TPGS hasbeen shown to increase the bioavailability of cyclosporin after livertransplants in children (see Sokol et. al.) Vitamin E TPGS may increasebioavailability by inhibiting cytochrome P450 drug metabolism in thegut. Vitamin E TPGS may inhibit the production of cyclosporin inducedrenal cytochrome P-450 and subsequent reduction in the formation ofoxygenated metabolites. Or, Vitamin E TPGS may inhibit P-gp-controlledback transport to increase the net transport of drugs through theenterocyte layer of the gut, causing an increase in the bioavailabilityof a coadministered drug. In addition, Vitamin E TPGS is a knownantioxidant. And, Vitamin E TPGS may play a role in arachidonic acidmetabolism by influencing prostaglandin formation by inhibiting thearachidonic acid release and enzyme activity of lipoxegenase. By thisprocess, Vitamin E TPGS may inhibit thrombocyte aggregation, and thusmay decrease the nephrotoxic effect of ISA_(TX)247 (15). Or, ISA_(TX)247may itself be more bioavailable and less toxic than cyclosporin A andother related compounds. Any one of these mechanisms, any combination ofthese mechanisms, or an unknown mechanism may contribute to theincreased bioavailability and decreased toxicity of the formulationsdisclosed herein.

Active Ingredient

The preferred active ingredient of the formulations of the presentinvention is an analog of cyclosporin, ISA_(Tx)247. ISA_(Tx)247 may bepresent in the formulation as a mixture of E- and Z-isomer or as asingle E- or Z-isomer. The structural formulas of the ISA_(TX)247isomers are as follows:

The present formulations are composed of the drug ISA 247, a syntheticor vegetable oil, preferably MCT (medium chain triglycerides) oilemulsified with a synthetic co-emulsifier such as a non-ionic surfactantthat has a polyoxyethylated moiety, preferably polyoxyethylated VitaminE. The formulated content can be adjusted for pH and isotonicity asneeded. These formulations can also include biocompatible polymers suchas poly(vinyl pyrrolidone) (PVP), poly vinyl alcohol (PVA) orpolyethylene glycol (PEG) and other polymers as protective colloids andsuspension or bulking agents, excipients, binders and/or carriers, asappropriate.

Synthesis of Mixtures of the (E) and (Z)-Isomers of ISA_(TX)247 Via theWittig Reaction

Wittig reaction pathways exemplified herein are identified by thereference numeral 31 in FIG. 3. Method 1 proceeds through the bromineintermediate acetyl-η-bromocyclosporin 41, whereas method 2 utilizes thealdehyde acetyl cyclosporin A aldehyde 51 as a starting point. Theexemplary methods described below utilize a Wittig reaction to introducean alkene functionality with a mixture of stereochemical configurations.

The Wittig reactions used in the exemplary embodiments disclosed hereinto synthesize mixtures of the (E) and (Z)-isomers of ISA_(Tx)247 mayoptionally be carried out in the presence of a lithium halide. Thepresence of lithium halides in Wittig reactions is well known to have aneffect on the ratio of geometrical isomers produced, and therefore theaddition of such a compound can aid in producing a desired mixture ofthe (E) and (Z)-isomers of ISA 247.

Method 1

In one embodiment of the present invention, a mixture of (E) and(Z)-isomers of ISA_(Tx)247 is prepared as shown in FIG. 4. The use ofthe wavy-lined representation in FIG. 4 (see especially compounds 43 and44) is meant to denote that the exemplary reaction sequence produces amixture of (E) and (Z)-isomers. In one embodiment the percentage ratioof the (E) to (Z)-isomers produced ranges from about 10 to 90 percent ofthe (E)-isomer to about 90 to 10 percent of the (Z)-isomer, but theseranges are only exemplary, and many other ranges are possible. Forexample, the mixture may contain from about 15 to 85 percent by weightof the (E)-isomer and about 85 to 15 percent of the (Z)-isomer. In otherembodiments, the mixture contains about 25 to 75 percent by weight ofthe (E)-isomer and about 75 to 25 percent by weight of the (Z)-isomer;about 35 to 65 percent by weight of the (E)-isomer and about 65 to 35percent by weight of the (Z)-isomer; and about 45 to 55 percent byweight of the (E)-isomer and about 55 to 45 percent of the (Z)-isomer.In still another embodiment, the isomeric mixture is an ISA 247 mixturewhich comprises about 45 to 50 percent by weight of the (E)-isomer andabout 50 to 55 percent by weight of the (Z)-isomer. These percentages byweight are based on the total weight of the composition, and it will beunderstood that the sum of the weight percent of the (E) isomer and the(Z) isomer is 100 weight percent. In other words, a mixture mightcontain 65 percent by weight of the (E)-isomer and 35 percent by weightof the (Z)-isomer, or vice versa.

Referring to FIG. 4, the terminal η-carbon of the side chain of the1-amino acid residue of acetyl-cyclosporin A is brominated in the nextstep of the reaction by refluxing acetyl cyclosporin A 35 withN-bromosuccinimide and azo-bis-isobutyronitrile in a solvent such ascarbon tetrachloride, producing the intermediateacetyl-η-bromocyclosporin A 41. N-bromosuccinimide is a reagent that isoften used to replace allylic hydrogens with bromine, and it is believedto do so via a free radical mechanism. The preparation of theintermediate 41 was essentially described by M. K. Eberle and F.Nuninger in “Synthesis of the Main Metabolite (OL-17) of Cyclosporin A,”J. Org. Chem., Vol. 57, pp. 2689-2691 (1992).

The novel intermediate triphenylphosphonium bromide of acetylcyclosporin A 42 may be prepared from acetyl-η-bromocyclosporin A 41 byheating the latter compound with triphenylphosphine in a solvent such astoluene.

The novel intermediate 42, and others like it, are contemplated to bekey intermediates in the synthesis of a plurality of cyclosporin Aanalogs that contain a conjugated diene system in the 1-amino acidresidue. For example, in addition to triphenylphosphine, compounds suchas triarylphosphines, trialkylphosphines, arylalkylphosphines, andtriarylarsines may be reacted with acetyl-η-bromocyclosporin A 41 toprepare other activated compounds similar to 42.

Referring again to FIG. 4, a mixture of the (E) and (Z)-isomers ofacetyl-1,3-diene 43 may be prepared by stirring the triphenylphosphoniumbromide of acetyl cyclosporin A 42 with an excess of formaldehyde intoluene at room temperature. Following addition of the formaldehyde, abase such as sodium hydroxide is added dropwise, and the isomericmixture of dienes is extracted with ethyl acetate.

Numerous organic chemistry textbooks describe the Wittig reaction. Onedescription in particular is provided by J. McMurry, Organic Chemistry,5^(th) Ed. (Brooks/Cole, Pacific Grove, 2000), pp. 780-783. A Wittigreaction may be used to convert a ketone or an aldehyde to an alkene. Insuch a process, a phosphorus ylide, also called a phosphorane, may bereacted with the aldehyde or ketone to give a dipolar intermediatecalled a betaine. Typically the betaine intermediate is not isolated;rather, it spontaneously decomposes through a four-membered ring toyield an alkene and triphenylphosphine oxide. The net result is areplacement of the carbonyl oxygen atom by the R₂C=group originallybonded to the phosphorus.

It will be appreciated by those skilled in the art that a wide varietyof reagents may be substituted for the exemplary Wittig reactionreagents cited above. For example, numerous alkyl, aryl, aldehyde, andketone compounds may be substituted for formaldehyde to prepare a vastnumber of cyclosporin derivatives. Applicants have carried out the abovesynthesis with formaldehyde, and in place of formaldehyde, compoundssuch as acetaldehyde, deuterated formaldehyde, deuterated acetaldehyde,2-chlorobenzaldehyde, benzaldehyde, and butyraldehyde. Such Wittigreactions may be carried out with compounds other thantriphenylphosphonium derivatives, such as triarylphosphines,trialkylphosphines, arylalkylphosphines and triarylarsines. Instead ofusing sodium hydroxide, various other bases such as sodium carbonate,butyllithium, hexyllithium, sodium amide, lithium hindered bases such aslithium diisopropylamide, and alkali metal alkoxides may be used. Inaddition to varying these reagents, the reaction may be conducted invarious organic solvents or mixtures of organic solvents and water, inthe presence of various salts, particularly lithium halides, and atvarying temperatures. All of the factors listed above can reasonably beselected by one of ordinary skill in the art to have the desired effecton the stereochemistry of the formed double bond; i.e., the desiredeffect on the ratio of the cis to trans-isomers.

In a final step of this synthesis, the protecting group on the p-carbonis removed using the following procedure. The mixture ofacetyl-(E)-1,3-diene and acetyl-(Z)-1,3-diene 43 is dissolved inmethanol, and then water is added. A base such as potassium carbonate isadded, and the reaction mixture stirred at room temperature. Bases otherthan potassium carbonate that may be used include sodium hydroxide,sodium carbonate, sodium alkoxide, and potassium alkoxide. Ethyl acetateis then used to extract the final product mixture of (E) and (Z)-isomersof ISA_(Tx)247 44.

Method 2

In an alternative reaction pathway for synthesizing a mixture of (E) and(Z)-isomers of ISA_(Tx)247 via a Wittig reaction strategy, a four stepsynthetic pathway may be employed as follows: 1) protection of theβ-alcohol, as in method 1, 2) oxidation of the acetyl-cyclosporin Aproduced from the first step to produce an aldehyde; 3) a Wittigreaction; and 4) de-acetylation of the Wittig reaction product, orequivalently, hydrolysis of the acetate ester to retrieve the alcohol.This reaction sequence is illustrated in FIG. 5.

This synthetic pathway begins in a manner similar to the Wittig reactionpathway of FIG. 4 in that the first step protects the β-alcohol with anacetate ester group. The two pathways differ from here on, however, inthat the next step of method 2 converts acetyl-cyclosporin A 35 to analdehyde, acetyl cyclosporin A aldehyde 51. This reaction uses anoxidizing agent sufficiently strong to cleave a C═C bond to produce twofragments. Alkene cleavage is known in the art. Ozone is perhaps themost commonly used double bond cleavage reagent, but other oxidizingreagents such as potassium permanganate (KMnO₄) or osmium tetroxide cancause double bond cleavage as well.

The use of ruthenium based oxidizing agents has been discussed by H. J.Carlsen et al. in “A Greatly Improved Procedure for Ruthenium TetroxideCatalyzed Oxidations of Organic Compounds,” J. Org. Chem., Vol. 46, No.19, pp 3736-3738 (1981). Carlsen et al. teach that, historically, theexpense of ruthenium metal provided an incentive for the development ofcatalytic procedures, the most popular of which used periodate orhypochlorite as stoichiometric oxidants. These investigators found aloss of catalytic activity during the course of the reaction with theconventional use of ruthenium which they postulated to be due to thepresence of carboxylic acids. The addition of nitriles to the reactionmixture, especially acetonitrile, was found to significantly enhance therate and extent of the oxidative cleavage of alkenes in a CCl₄/H₂O/IO₄″system.

According to one embodiment of the present invention, acetyl cyclosporinA aldehyde 51 may be produced from acetyl cyclosporin A 35 by dissolvingit in a mixture of acetonitrile and water, and then adding first sodiumperiodate and then ruthenium chloride hydrate. The aldehyde 51 may beextracted with ethyl acetate. It should be noted that the synthesis ofthe aldehyde 51 by this oxidative cleavage strategy is important to manyof the stereoselective pathways.

The third step of method 2 involves converting the aldehyde 51 to amixture of (E) and (Z) dienes via a Wittig reaction, in a similarfashion to that of method 1. As in method 1, a phosphorus ylide adds tothe aldehyde to yield a betaine (which is not isolated), with the netresult that the carbonyl oxygen atom of the aldehyde is replaced by theR₂C=group originally bonded to phosphorus. Again, such Wittig reactionsmay be carried out with phosphorus containing compounds other thantriphenylphosphonium derivatives, such as triarylphosphines,trialkylphosphines, arylalkylphosphines and triarylarsines, at varioustemperatures, and using a variety of basic solutions and solvents or theaddition of various inorganic salts may be used to influence thestereochemistry of the newly formed double bond.

In one embodiment, acetyl cyclosporin A aldehyde 53 is dissolved intoluene, to which is added a base such as sodium hydroxide in water.Allyl triphenylphosphonium bromide 52 is then added, and the reactionstirred for some time. Workup of the product mixture of acetyl (E) and(Z)-1,3-dienes 53 involves extraction with hexane and/or ethyl acetate,where the term “workup” is intended to mean the process of extractingand/or isolating reaction products from a mixture of reactants,products, solvent, etc.

In a final step of method 2, similar to the final step of method 1, theacetate ester group protecting the alcohol at the β-carbon position isremoved with potassium carbonate, yielding a mixture of (E) and (Z)isomers of ISA_(TX)247 54. Bases other than potassium carbonate that maybe used to remove the protecting group include sodium hydroxide, sodiumcarbonate, sodium alkoxide, and potassium alkoxide.

Vitamin E TPGS

Tocopheryls are used in the formulations as antioxidants. D-a-tocopherylpolyethylene glycol 1000 succinate (Vitamin E TPGS) (Eastman Kodak,Rochester, N.Y.) is a water soluble derivative of Vitamin E, and has adual nature of hydrophilicity and lipophilicity and has been used as anantioxidant or as a preservative in formulations known in the art. Whenused as an antioxidant, tocopheryl or Vitamin E TPGS may be present infrom about 0.5% to about 1%, and not more than 5% percent by weight.

Vitamin E TPGS is miscible in water and forms solutions with water atconcentrations up to approximately 20% weight, beyond which liquidcrystalline phases may form. Vitamin E TPGS has a high degradationtemperature and good thermal stability. Micelles are formed at 0.02percent weight. When Vitamin E TPGS concentration is above 20 percentweight, higher viscosity liquid crystalline phases form. With increasingVitamin E TPGS concentration in water, more complex liquid crystallinephases evolve from isotropic globular micellar to isotropic cylindricalmicellar and hexagonal, hexagonal, mixed hexagonal and reversedhexagonal, reversed globular micellar, and to the lamellar phase (13).In these formulations, Vitamin E TPGS is useful as a surface activeagent or as an emulsifier. It may also be used in complex formulationsthat include a number of other excipients that function as solvent,binder, and filler.

In addition, the Vitamin E TPGS in the present invention may be presentnot only as an emulsifying agent and adjuvant, but also to inhibit thealteration of the oil, preventing them from becoming rancid.

Oil Component

The oil component of the present formulations may be a vegetable oil, asynthetic oil, an oil substitute such as triacetin, a mineral oil or amedium chain triglyceride (MCT) oil, i.e., a triglyceride oil in whichthe carbohydrate chain has 8-10 carbon atoms. Preferably, the oilcomponent is MCT oil.

MCT oil has many advantages over vegetable oil, including: 1) a lowersusceptibility to oxidation; 2) a specific density of about 0.94-0.95which is higher than that of vegetable oil and which is close to that ofwater thus facilitating the obtaining of a stable microemulsion; 3) itis less hydrophobic than vegetable oil and therefore enables theachievement of higher concentrations of the drug dissolved therein; and4) it has a lower viscosity which enables obtaining a higherconcentration of the oily phase in the composition without substantiallyincreasing its viscosity (15).

MCT oil is available commercially. Examples of MCT oils contemplated bythe present invention include, but are not limited to, TCR™ (trade nameof Societe Industrielle des Oleagineaux, France, for a mixture oftriglycerides wherein about 95% of the fatty acid chains have 8 or 10carbons) and MIGLYOL 812™ (trade name of Dynamit Nobel, Sweden, for amixed triester of glycerine and of caprylic and capric acids). Isopropylmyristate is another commercially available oil useful in formulationsof the present invention.

Examples of vegetable oils contemplated by the present inventioninclude, but are not limited to, soybean oil, cotton seed oil, oliveoil, sesame oil and castor oil. Mineral oils include, but are notlimited to, natural hydrocarbons or their synthetic analogs. Oily fattyacids, such as oleic acid and linoleic acid, fatty alcohols, such asoleyl alcohol, and fatty esters, such as sorbitan monooleate andhydrophobic sucrose esters, may be used as the oil component, althoughthese are not as preferred as the other oils mentioned herein. Otherlipids which may be used in the formulations of the present inventioninclude, but are not limited to, synthetic and semi-synthetic mono-, di-and/or triglycerides, triglycerides prepared by solvent or thermalfractionation of natural, synthetic or semisynthetic triglycerides, andtriglycerides prepared by interesterification and/or directed or randomrearrangement.

Emulsifiers

The formulations of the present invention contain at least oneemulsifier. Preferably the emulsifier or surfactant is a non-ioniclipophilic solvent or a non-ionic amphiphilic solvent; the emulsifiermay be a Tween, such as Tween 40, Tween 80, or the like. However, anysurfactant capable of forming and microemulsion preconcentrate may beused. Some examples are given below.

The surfactant component may comprise amphiphilic, hydrophilic orlipophilic surfactants, or mixtures thereof. Especially preferred arenon-ionic hydrophilic and non-ionic lipophilic surfactants. Examples ofsuitable hydrophilic surfactants for use as surfactant components arereaction products of natural or hydrogenated vegetable oils and ethyleneglycol. Such products may be obtained in known manner, e.g. by reactionof a natural or hydrogenated castor oil or fractions thereof withethylene oxide, with optional removal of free polyethyleneglycolcomponents from the product. Also suitable are various tensides, orsurfactants, available under the trade name Cremophor™. Particularlysuitable is Cremophor™ RH 40 and Cremophor™ EL. Also suitable for use inthis category are the various tensides available under the trade nameNikkol™.

Other examples include polyoxyethylene-sorbitan-fatty acid esters, e.g.,mono- and trilauryl, pa′lmityl, stearyl and oleyl esters e.g., Tweens,including polyoxyethylene(20)sorbitanmonolaurate [Tween® 20],polyoxyethylene(20)sorbitanmonopalmitate [Tween® 40],polyoxyethylene(20) sorbitanmonostearate [Tween® 60],polyoxyethylene(20)sorbitanmonooleate [Tween® 80],polyoxyethylene(20)sorbitantristearate [Tween® 65],polyoxyethylene(20)sorbitantrioleate [Tween 85],polyoxyethylene(4)sorbitanmonolaurate [Tween® 21],polyoxyethylene(4)sorbitanmonostearate [Tween® 61], andpolyoxyethylene(5) sorbitanmonooleate [Tween® 81]. Especially preferredproducts of this class for use in the compositions of the invention areTween 40 and Tween 80, polyoxyethylene fatty acid esters, such aspolyoxyethylene stearic acid esters, polyoxyethylene-polyoxypropyleneco-polymers, polyoxyethylene-polyoxypropylene block co-polymers, e.g. ofthe type known and commercially available under the trade namePoloxamer™, dioctylsuccinate, dioctylsodiumsulfosuccinate,di-2-ethylhexyl-succinate or sodium lauryl sulfate, and phospholipids,in particular, lecithins. Lecithins suitable for use in the compositionsof the invention include, but are not limited to, soya bean lecithins.Other suitable products include Cremophor™, Nikkol™, glycolated naturalor hydrogenated vegetable oils, and caprylic-capric acid diester.Propylene glycol mono- and di-fatty acid esters such as propylene glycoldicaprylate, propylene glycol dilaurate, propylene glycolhydroxystearate, propylene glycol isostearate, propylene glycol laurate,propylene glycol ricinoleate, propylene glycol stearate are alsosuitable.

The formulations of the present invention may further comprise apharmaceutically acceptable surfactant, colloid, suspension, bulkingagent, excipient, carrier, tenside or cotenside.

B. Preferred Formulations of the Present Invention

Embodiments of the present invention are preferably stable microemulsionpreconcentrates which provide sufficient drug bioavailability and/orreduced toxicity. These formulations may have superior ability toincrease drug bioavailability and to reduce adverse effects associatedwith the administration of cyclosporin and its analogs. Microemulsionformulations allow for delivery of a drug in smaller particles. Deliveryof a unit dosage of a drug in smaller particles allows for increasedsurface area and presumably higher absorption and betterbioavailability. Microemulsions are known in the art. However,microemulsions using the components of the present formulation areformed by the judicious choice of ratios of ethanol, Vitamin E TPGS, oilcomponent, and emulsifier which are outside the ranges of previous knownformulations for active agents similar to ISA_(Tx)247. Compositionscontaining less than 5% of ethanol, or greater than 50% of Vitamin ETPGS form solid microemulsion preconcentrates at room temperature, whichis a less preferred embodiment of the formulations of this invention.

The relative proportion of ingredients in the compositions of theinvention will, of course, vary considerably depending on the particulartype of composition concerned, e.g. whether it is a “microemulsionpreconcentrate”, microemulsion, regular emulsion, solution and so forth.The relative proportions will also vary, depending on the particularfunction of ingredients in the composition, for example, in the case ofa surfactant component of a “microemulsion preconcentrate”, on whetherthis is employed as a surfactant only or both a surfactant and aco-solvent. The relative proportions will also vary depending on theparticular ingredients employed and the desired physical characteristicsof the product composition. Determination of workable proportions in anyparticular instance will generally be within the capability of theskilled artisan. All indicated proportions and relative weight rangesdescribed below are accordingly to be understood as being indicative ofpreferred or individually inventive teachings only and not as notlimiting the invention in its broadest aspect. The microemulsions of thepresent inventions may be microemulsion preconcentrates. Preferredformulations of the present invention are as follows:

Formulation 1

a) a pharmaceutically effective amount of ISA_(TX)247;

b) Vitamin E TPGS;

c) MCT oil;

d) Tween 40; and

e) ethanol

in a ratio of ingredients of 0.5-1:4:2:2:1.

Formulation 2

a) a pharmaceutically effective amount of ISA_(TX)247;

b) Vitamin E TPGS;

c) MCT oil;

d) Tween 80; and

e) ethanol

in a ratio of ingredients of 0.5-1:2:4:2:1.

Formulation 3

a) a pharmaceutically effective amount of ISA_(TX)247;

b) MCT oil;

c) Tween 80;

d) triacetin; and

e) ethanol

in a ratio of ingredients of 0.5-1:5:3:1:1.

Formulation 4

a) a pharmaceutically effective amount of ISA_(TX)247;

b) Tween 80;

c) Vitamin E TPGS;

d) ethanol; and

e) isopropyl myristate

in a ratio of ingredients of 0.5-1:2:1:1:6.

Formulation 5

a) a pharmaceutically effective amount of ISA_(Tx)247;

b) Vitamin E TPGS;

c) MCT oil;

d) Tween 40; and

e) ethanol

in a ratio of ingredients of 0.5-1:4:2:2:1, with the formulation furthermixed with an aqueous medium to form a clear stable microemulsionsolution. Aqueous media may include, but are not limited to water, fruitjuices, such as apple juice, tea, milk and cocoa. Useful fruit juices,exclude grapefruit juice and preferably include apple juice. Theformulation is preferably dissolved into the aqueous media at a ratio ofabout 1 part formulation to about 10 to about 100 parts aqueous media.

The pharmaceutical formulations of the present invention preferablyallow for sufficient bioavailability. Preferably, the bioavailabllity ofISA_(Tx)247 is equal to or greater than 30%, more preferably thebioavailability is greater than 40%. The pharmaceutical formulations arepreferably in a form for oral administration, such as microemulsions, ormicroemulsion preconcentrates. The aqueous media is preferably one whichis palatable to a patient so that they will consume the oral formulationwith ease.

When administered to a patient in need thereof in an effective amount,the formulations of the present invention will produceimmunosuppression. Preferably, the amount of the pharmaceuticalformulation administered is about 0.1 to 10 mg/kg of body weight of thesubject per day, more preferably about 0.5 to 3 mg/kg of body weight ofthe subject per day. The formulation is preferably administered eitheronce a day or twice a day.

When properly administered, the formulations of the present inventionmay be used for immunosuppression including, for example, to preventorgan rejection or graft vs. host disease, and to treat diseases andconditions, in particular, autoimmune and inflammatory diseases andconditions. Examples of autoimmune and inflammatory diseasescontemplated herein include, but are not limited to, Hashimoto'sthyroiditis, pernicious anemia, Addison's disease, psoriasis, diabetes,rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis,Sjogren's syndrome, dermatomyositis, lupus erythematosus, multiplesclerosis, myasthenia gravis, Reiter's syndrome, arthritis (rheumatoidarthritis, arthritis chronica progrediente and arthritis deformans) andrheumatic diseases, autoimmune hematological disorder (hemolyticanaemia, aplastic anaemia, pure red cell anaemia and idiopathicthrombocytopaenia), systemic lupus erythematosus, polychondritis,sclerodoma, Wegener granulamatosis, dermatomyositis, chronic activehepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome,idiopathic sprue, autoimmune inflammatory bowel disease (ulcerativecolitis and Crohn's disease) endocrine opthalmopathy, Graves disease,sarcoidosis, primary billiary cirrhosis, juvenile diabetes (diabetesmellitus type I), uveitis (anterior and posterior), keratoconjunctivitissicca and vernal keratoconjunctivitis, interstitial lung fibrosis,psoriatic arthritis and glomerulonephritis.

The formulations of the present invention may also be used to treatanti-parasitic, or anti-protozoal diseases such as malaria,coccidiomycosis and schistosomiasis. They may also be used as an agentfor reversing or abrogating anti-neoplastic agent resistance in tumors.

The present invention also contemplates a formulation to be administeredparenterally (e.g., subcutaneously or intramuscularly). This formulationcomprises the following:

-   -   a) a pharmaceutically effective amount of ISA_(Tx)247;    -   b) Vitamin E TPGS;    -   c) MCT oil;    -   d) Tween 40; and    -   e) ethanol.

Preferably such a formulation is sterilized prior to administration.

C. Methodology

We have found that stable formulations of the cyclosporin analogISA_(Tx)247 can be prepared. The formulations of the present inventionmay be prepared in the following manner. However, the person skilled inthe art knows that any sequence of these steps is possible. In addition,the amounts of components employed in the following is as detailed aboveand the methods described below can readily convert one or more steps orcompletely inverse the process.

While only Formulation 5 details how the microemulsion preconcentrate isadded to an aqueous solution to form a microemulsion, any of themicroemulsion preconcentrate formulations may be converted tomicroemulsions by mixing with an aqueous media. Preferably, thepreconcentrate is mixed with the aqueous media at a ratio of 1 partpreconcentrate to 10-100 parts media. More preferably the aqueous mediais apple juice.

Formulation 1

First, the MCT oil is added to the Tween 40 to form a mixture at roomtemperature (20-25° C.). Next, ethanol is added to the mixture of MCToil and Tween 40. Next, Vitamin E TPGS is added to the mixture. Themixture is then incubated with stirring at 30-35° C. until clear. Then,the proper amount of ISA_(Tx)247 is added. Finally, the ingredients aremixed at 30-35° C. until the ISA_(Tx)247 is completely dissolved.

Formulation 2

First, the MCT oil is added to the Tween 80 to form a mixture at roomtemperature (20-25° C.). Next, ethanol is added to the mixture of MCToil and Tween 80. Next, Vitamin E TPGS is added to the mixture. Themixture is then incubated with stirring at 30-35° C. until clear. Then,the proper amount of ISA_(Tx)247 is added. Finally, the ingredients aremixed at 30-35° C. until the ISA_(Tx)247 is completely dissolved.

Formulation 3

First, the MCT oil is added to the Tween 80 to form a mixture at roomtemperature (20-25° C.). Next, ethanol is added to the mixture of MCToil and Tween 80. Next, triacetin is added to the mixture. The mixtureis then incubated with stirring at 30-35° C. until clear. Then, theproper amount of ISA_(Tx)247 is added. Finally, the ingredients aremixed at 30-35° C. until the ISA_(Tx)247 is completely dissolved.

Formulation 4

First, the MCT oil is added to the Tween 80 and mixed at roomtemperature (20-25° C.). Next, isopropyl myristate and ethanol are addedto the mixture at room temperature (20-25° C.). Then, the proper amountof ISA_(Tx)247 is added. Finally, the ingredients are mixed until theISA_(Tx)247 is completely dissolved at room temperature (20-25° C.).

Formulation 5

First, the MCT oil is added to the Tween 40 to form a mixture at roomtemperature (20-25° C.). Next, ethanol is added to the mixture of MCToil and Tween 40. Next, Vitamin E TPGS is added to the mixture. Themixture is then incubated with stirring at 30-35° C. until clear. Then,the proper amount of ISA_(Tx)247 is added. Finally, the ingredients aremixed at 30-35° C. until the ISA_(Tx)247 is completely dissolved. Theresulting preconcentrate is then mixed with aqueous media. Preferably,the preconcentrate is mixed with the aqueous media at a ratio of 1 partpreconcentrate to 10-100 parts media.

In preferred methodologies, each of the above formulations are preparedby first dissolving the ISA_(Tx)247 in ethanol. The rest of theingredients may be added in any order.

DISCLOSURE OF THE EXAMPLES OF THE INVENTION

In the examples below, the following abbreviations have the followingmeanings. If an abbreviation is not defined it has its generallyaccepted meaning

-   -   w/w=weight to weight    -   NF=National Formulary    -   NF/NP=National Formulary/National Pharmacopeia    -   USP=United States Pharmacopeia    -   mg=milligrams    -   mL=milliliters    -   kg=kilograms    -   ng=nanograms    -   nm=nanometers    -   T_(max)=Time of maximum concentration    -   C_(max)=maximum concentration    -   AUC=area under the curve    -   LCMS=Liquid Chromatography-Mass Spectrometry    -   MCT=Medium chain triglycerides    -   PK=Pharmacokinetics    -   PS=pig skin

Two drug products, an oral solution and a soft gelatin capsule, havebeen developed by Isotechnika, Inc. Both drug products contain asurfactant, ethanol, a lipophilic and/or an amphiphilic solvent asingredients. Preparation of each is described below.

Example 1 Preparation of ISA_(Tx)247 Oral Solution

The following ingredients make up the ISA_(Tx)247 drug product-oralsolution: ISA_(Tx)247, d-alpha Tocopheryl polyethylene glycol 1000succinate (vitamin E TPGS) NF, Medium Chain Triglycerides (MCT) Oil USP,Tween-40 NF, and 95% Ethanol USP.

TABLE 1 Composition of ISA_(TX)247 Oral Solution Unit and/or PercentageFormula Quantity per Batch Ingredient (and Test Strength: 50 Lot Lot LotStandard) mg/mL #39120228 #30010532 #30040517 d-alpha Tocopheryl 42.24%0.16205 kg 0.544 kg 0.768 kg polyethylene glycol 1000 succinate (vitaminE TPGS) NF Medium Chain 21.12% 0.08102 kg 0.272 kg 0.384 kgTriglycerides (MCT) Oil USP Tween-40 NF 21.12% 0.08102 kg 0.272 kg 0.384kg 95% Ethanol USP 10.56% 0.04051 kg 0.136 kg 0.192 kg ISA_(Tx)247 4.95%0.01900 kg 0.0637 kg  0.090 kg Total 99.99% 0.38360 kg 1.286 kg  L818 kg

Vitamin E TPGS was melted in a hot box and transferred to a liquiddispenser. The warm contents of each TPGS container were mixed for atleast one minute to ensure that it was a visually homogeneous liquid.When the temperature of the contents of each barrel was no less than 35°C., a Groen vessel at the required temperature 37.5° C.±2.5° C. wasprepared.

The required amount of ingredients Vitamin E TPGS, MCT Oil, Tween 40 and95% ethanol were weighed in separate vessels properly labeled foridentification. The Vitamin E TPGS was immediately transferred into theGroen vessel. The MCT Oil, Tween 40, and ethanol were transferred in astepwise fashion quantitatively to the vessel containing pre-weighedVitamin E TPGS. The vessel was securely covered to reduce ethanolevaporation and mixed at 37.5±2.5° C. for 15-20 minutes after theaddition of ethanol.

The top and bottom samples were observed using sterile glass disposablepipettes. The pipette contents were expected to be homogeneous and clearwhen viewed.

The mixer was set so that a slight vortex was achieved. The containerwas covered to reduce ethanol evaporation during mixing. ISATx247 wasadded to the continuously mixing blend. Then the container was sealed.The mixture was checked every half hour until the powder had completelydissolved.

Example 2 Preparation of ISA_(Tx)247 Gelatin Capsule

The present invention also contemplates a soft gelatin capsule. Thiscapsule contains ISATx247 and the same non-medicinal ingredients as theoral solution encapsulated in gelatin type 2D PS(NF/NP) capsulescontaining gelatin, glycerin, water, and sorbitol.

The ISA_(Tx)247 gelatin capsule was prepared by encapsulating theISA_(Tx)247 oral solution (50 mg/mL) comprised of ISA_(Tx)247, Vitamin ETPGS NF, medium chain triglycerides (MCT) oil USP, Tween-40 NF, and 95%ethanol USP, into gelatin type 2D PS(NF/NP) capsules. The gelatin type2D PS(NF/NP) capsules was prepared using gelatin NF (pig skin), glycerinUSP, purified water USP, and 76% sorbitol special.

Example 3 Stability/Shelf-Life Measurements

The Table below shows stability data, as measured by well known LCMStechniques, for Formulation 1 in both bottled (stored at 30° C.) andsoft-gel capsule forms (stored at room temperature). The formulationsare stable under normal storage conditions.

TABLE 2 ISA_(TX)247 Concentration (mg/mL) 0 Months 6 Months 24 MonthsBottled 57.1 ± 0.5 56.7 ± 0.7 58.0 ± 0.6  Formulation Soft-gel Capsules41.1 ± 0.6 42.4 ± 1.0 41.4 ± 0.9 ^(A) ^(A) 18 month measurement forsoft-gel capsules

Example 4 Comparison of Pharmacokinetic (PK) Profiles

The formulations indicated below were given to Sprague Dawley rats byoral gavage and blood levels of ISA_(TX)247 were monitored at regularintervals over time to generate a concentration versus time curve (i.e.,pharmacokinetic profile). Below is a comparison of the PK profiles ofvarious ISA_(Tx)247 formulations, including those disclosed herein.While group number 2 shows good bioavailability in the rat model, asshown in Table 3, these results may vary substantially in dog or humanmodels (see table 4).

TABLE 3 PK profiles of various ISA_(Tx)247 formulations BioavailabilityRoute of relative to Admin. Group T_(max) C_(max) Half-life AUC Group 1Oral 1 2 49 7.3 559 +0 Oral 2 4 75 6.1 1150  +106 Oral 3 4 36 18.6 491−12 Oral 4 4 68 7.4 885 +58 Oral 5 2 104 7.1 1298  +132 Oral 6 4 57 7.7 750′ +34 T_(max) = Time (hours) to maximum blood level of ISA_(Tx)247C_(max) = Maximum blood level (ng/mL) of ISA_(Tx)247 Half-life = Time(hours) to reach half maximal ISA_(Tx)247 blood level AUC = Area underthe curve. (Related to bioavailability of drug.)

Group 1:

-   -   VitE-TPGS/MCT Oil/Tween 40/ethanol: 4/2/2/1 (w/w/w/w).        (Disclosed herein as Formulation 1).

Group 2:

-   -   VitE-TPGS/MCT Oil/Tween 807 ethanol: 2/4/2/1 (w/w/w/w)        (Disclosed herein as Formulation 2).

Group 3:

-   -   MCT Oil/Tween 807 Triacetin/ethanol: 5/3/1/1 (w/w/w/w)        (Disclosed herein as Formulation 3).

Group 4:

-   -   Tween 80/VitE-TPGS/ethanol/isopropyl myristate: 2/1/1/6        (w/w/w/w) (Disclosed herein as Formulation 4).

Group 5 (comparative example):

-   -   Vit E/propylene glycol/Maisine/Chremophor RH 40/ethanol:        3/1.2/5/6.2/2 (w/w/w/w).

Group 6:

-   -   This is the formulation diluted 1:65 in apple juice to form a        microemulsion. (Disclosed herein as Formulation 5).

Example 5 Preparation of Microemulsions

Over 100 ratio permutations of formulation components were tested formicroemulsion formation (defined as less than 0.1 and 0.01 absorbance at485 and 900 nm, respectively). Formulations tested included ratios ofcomponents from:

-   -   5-10% ISA_(Tx)247    -   0-50% Vitamin E-TPGS    -   0-50% Tween 40 and/or Tween 80    -   0-50% MCT Oil    -   5-15% Ethanol.        Less than 20% of the formulations tested produced        microemulsions. The microemulsion formation is relatively        independent of the ratio of either Vitamin E-TPGS or ethanol.        However, formulations with ratio's of Vitamin E-TPGS greater        than 50%, and ethanol less than 5%, result in preconcentrates        that are frozen at room temperature, which are less preferred.        FIG. 1 shows a table for the formation of microemulsion        preconcentrates as a function of excipient ratio for Tween 40        and MCT Oil.

Example 6 Comparison of Pharmacokinetic TPKI Profiles in Dogs

FIG. 2 shows a graph providing a comparison of pharmacokinetic (PK)profiles for our ISATx247 microemulsion in comparison with Neoral® inBeagle dogs. The data is also tabulated below. The formulations weregiven to Beagle dogs (n=6) by oral gavage and blood levels of ISATx247were monitored at regular intervals over time to generate aconcentration versus time curve (i.e., pharmacokinetic profile).Specifically, dogs were given 2 mL of either formulation (100 mg/mL) byoral gavage. Blood was withdrawn at T=0, 0.5, 1, 2.5, 5, 7.5, 12 and 24hours post-dose and analyzed by Liquid Chromatography-Mass Spectrometry(LCMS).

TABLE 4 Summary of Results Route of Administration Group T_(max) C_(max)AUC Oral 1 2 H 1439 ± 378 8290 Oral 2 2 H 2158 ± 677 11460 Tmax = Time(hours) to maximum blood level of ISA_(TX)247 Cmax = Maximum blood level(ng/ml) of ISA_(TX) 247 AUC = Area under the curve. (Related tobioavailability of drug.)

Group 1: Formulation 1: VitE-TPGS/MCT Oil/Tween 407 ethanol: 4/2/2/1(w/w/w/w).

Group 2: Neoral®

Although these data indicate a slightly lower bioavailability of theISA_(Tx)247 formulation, relative to Neoral, it is substantially greaterthan the bioavailability provided by Sandimmune®. (Data not shown)Neoral® is generally reported to be about 40% bioavailable. As Table 4illustrates, the bioavailability of Group number 1 is comparable to thatof Neoral®.

These data indicate that the formulations of the present invention mayhave bioavailability comparable to other formulations of cyclosporin andmay reduce adverse effects associated with the administration ofcyclosporin.

Example 7 Pharmacokinetic and Toxicokinetic Properties of ISA_(Tx)247and Cyclosporin a

The pharmacokinetic and toxicokinetic parameters of ISA_(Tx)247 (45-50%of E-isomer and 50-55% of Z-isomer) and cyclosporine A were tested in arabbit model. The rabbit has also been used as a model to studycyclosporine A nephrotoxicity, but far less frequently than the rat.Studies have found that cyclosporine A administered to the rabbit causesstructural and functional changes at a dose not only lower than has beenpreviously reported in other animal models, but also within at least theupper level of the therapeutic range in humans (Thliveris et al., 1991,1994). Also, the finding of interstitial fibrosis and arteriolopathy, inaddition to the cytological changes in the tubules, suggests that therabbit is a more appropriate model to study nephrotoxicity, since thesestructural entities are hallmarks of nephrotoxicity observed in humans.ISA_(Tx)247 was administered intravenously (i.v.) for the first 7 daysand subcutaneously (s.c.) for an additional 23 days according to thefollowing schedule.

TABLE 5 The Dose Administration Schedule for the Investigation of thePharmacokinetic and Toxicokinetic Properties of ISA_(TX)247 in theRabbit Model Days 1-7: Days 8-30: i.v. Dose s.c. Dose Numberof AnimalsTreatment Group (mg/kg) (mg/kg) Males Females 1. Vehicle Control 0 0 4 42. Cyclosporine A 10 10 6 6 3. Low-Dose 5 5 0 2 4. Medium-Dose 10 10 4 45. High-Dose 15 15 4 4

Pathogen free rabbits (SPF) were used to ensure any renal changesobserved were due to the effect of ISA_(Tx)247 and not due to pathogens.On Days 1 and 7, blood samples were collected prior to drugadministration and at 0.5, 1, 2, 4, 8, 12, 18, and 24 hours post-dose togenerate a pharmacokinetic profile. Other evaluated parameters includedclinical observations, body weight, food consumption, hematology,clinical chemistry, gross pathology, and histopathological examinationof selected tissues/organs.

Blood samples were analyzed via high performance liquid chromatographycoupled with mass spectrometry (LCMS). Table 6 below summarizes theaverage pharmacokinetic parameters in rabbits that received 10 mg/kg ofcyclosporine A or ISA_(Tx)247.

TABLE 6 Pharmacokinetic Parameters of Intravenously AdministeredCyclosporine A and ISA_(TX)247 in Male Rabbits Receiving 10 mg/kg/day.Results expressed as mean ± SD Measured Cyclosporine A ISA_(TX)247Parameter Day 1 Day 7 Day 1 Day 7 T_(max) (hours) 0.5 0.5 0.5 0.5C_(max) (@g/L) 1954 ± 320  2171 ± 612  19 15 ± 149  1959 ± 470  t½(hours) 7.4 ± 2.8 9.0 ± 4.0 7.4 ± 1.7 9.2 ± 1.1 AUC (@g@hr/L) 6697 ±1717 6685 ± 1247 5659 ± 1309 5697 ± 1373

There were no statistically significantly differences between thepharmacokinetic parameters of Cyclosporine A and ISA_(Tx)247 in malerabbits receiving 10 mg/kg/day. The pharmacokinetic parameters of ISA247 in female rabbits receiving the same dose were not significantlydifferent from that observed in the male rabbits, with the exception ofmaximum concentration on Day 7.

No significant changes were noted in the hematological parameters ofrabbits receiving a vehicle control, Cyclosporine A, or ISA_(TX)247. Adifference was noted in the creatinine levels in the various groups overthe course of the study, as is shown in Table 7 below. These differencesindicated that Cyclosporine A had a significantly greater negativeeffect on the kidneys than either the vehicle control or ISA_(TX)247. Itshould be noted that even at a 50% higher dose, 15 mg/kg/day, ascompared to 10 mg/kg/day Cyclosporine A, ISA_(Tx)247 did not result inany significant increase in serum creatinine levels.

TABLE 7 Percent Change in Serum Creatinine Levels Over Baseline in MaleRabbits Receiving Vehicle, Cyclosporine A, or ISA_(TX)247 for 30 DaysTreatment Group Day 15 Day 30 Vehicle  +6%  −3% Cyclosporine A (10mg/kg) +22% +33% ISA_(TX)247 (10 mg/kg)  +1% +10% ISA_(TX)247 (15 mg/kg)−19% −11%

Examination of organs in all rabbits receiving the vehicle control, 10mg/kg cyclosporine A, 5 mg/kg ISA_(Tx)247, or 10 mg/kg ISA_(Tx)247revealed no significant abnormalities. This was especially true for thekidneys, in which no evidence of interstitial fibrosis, normally seen incyclosporine A-treated animals (Thliveris et al., 1991, 1994) was noted.In male rabbits that received 15 mg/kg ISA_(D)(247, a decrease inspermatogenesis was noted. No changes were noted in the 3 female rabbitsthat completed the study at this dose of 15 mg/kg ISA_(Tx)247.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

All references discussed above are herein incorporated by reference intheir entirety.

What is claimed:
 1. A pharmaceutical composition comprising: a) apharmaceutically effective amount of ISA_(Tx)247; b) Tween 80; c)Vitamin E TPGS; d) ethanol; and e) isopropyl myristate; wherein thepharmaceutical composition is a microemulsion or a microemulsionpreconcentrate.
 2. The pharmaceutical composition of claim 1 whichcomprises from about 5% to about 10% by weight of ISA_(Tx)247, fromabout 20% to about 50% by weight of Vitamin E TPGS, from about 5% toabout 55% by weight of isopropyl myristate, from about 5% to about 50%by weight of Tween 80, and about 5% to about 15% by weight of ethanol.3. The pharmaceutical composition of claim 2, wherein the composition isa liquid microemulsion preconcentrate at room temperature.
 4. Thepharmaceutical composition of claim 1, wherein the ratio by weight ofISA_(Tx)247:Tween 80:Vitamin E TPGS:ethanol:isopropyl myristate is about0.5-1:2:1:1:6.
 5. A method of producing immunosuppression, comprisingadministering to a subject in need thereof an effective amount of thepharmaceutical composition of claim
 1. 6. A method of preparing thepharmaceutical composition of claim 1, comprising mixing the isopropylmyristate, the Tween 80, the ethanol, the Vitamin E TPGS, and thepharmaceutically effective amount of ISA_(Tx)247 until the ISA_(Tx)247is completely dissolved.